Fig. 3

Effects of AGEs on eNOS protein levels and NOS activity in HCAECs. a Western blot analysis. Cells were treated with 50, 100 or 200  μg/ml AGEs for 24 h. Representative bands of eNOS and β-actin staining and quantitation of band density ratios (eNOS and β-actin) Full-length blots are presented. *P < 0.05 compare with control, n = 3 experiments. Data are means and SE of multiple experiments (n). b NOS activity. Cells were treated withs AGEs for 24 h. The NOS activity was determined by a commercial eNOS fluorimetric assay kit. c Cellular NO levels. Cells were treated with AGEs (100 μg/l) for 24 h and cellular NO levels were determined by DAF-FM DA (10 μM) staining and flow cytometric analysis. d Nitric oxide (NO) levels in the supernatant of cell culture (Griess assay). Cells were treated with AGEs (100 μg/ml) in the presence or absence of LY-83,583 (3 μM) or l-NAME (100 μM) for 24 h. Basal and bradykinin-stimulated levels of NO-derived nitrite in the culture supernatant were detected. * P < 0.05, compare with control, n = 3 experiments. Data are means and SE of multiple experiments (n)